Indian hedgehog (Ihh) has multiple functions during skeletogenesis. Mice lacking the Ihh gene exhibit severe skeletal abnormalities, including markedly reduced chondrocyte proliferation and abnormal maturation, with absence of mature osteoblasts. Since Ihh and its receptor, smoothened (smo), are expressed in chondrocytes as well as in osteoblasts, current animal models do not provide sufficient information whether Ihh has a direct effect on osteoblasts, or whether the effects on bone are indirectly mediated through chondrocytes during endochondral ossification. In this grant application we propose to selectively ablate, in vivo, the Ihh gene from collagen type II expressing cells, pre- and postnatally, and from a subset of collagen type X expressing cells. In addition, we will delete smo selectively from chondrocytes and compare the phenotypes. This will allow us to determine the specific role of chondrocyte-derived Ihh on endochondral bone formation. We plan to use animals that we generated and cross bred with col2alpha1-cre, col2alpha1-creER*, and col10alpha1-cre mice, obtained from our collaborators. In Specific Aim 1 we propose to selectively delete either the Ihh or the smo gene from chondrocytes, before birth, and to analyze and compare the mutant phenotypes to define the role of Ihh during the process of endochondral bone formation. In Specific Aim 2 we will analyze mice in which the Ihh gene will be selectively deleted from chondrocytes after birth. We will take advantage of a tamoxifen-inducible ere that is under the control of the collagen type II promoter to determine the acute effects of Ihh on established endochondral bone. In Specific Aim 3 a hypomorph mouse model will be generated, in which only a subset of chondrocytes in the growth plate will be deleted for the expression of Ihh. For this purpose, collagen type X cre knock-in mice will be bred with floxed Ihh animals. Using this model we will be able to detect the response of chondrocytes to reduced Ihh expression. We expect a less severe phenotype in these mice, and will perform a comparative analysis between animals lacking the Ihh gene in collagen type II and collagen type X expressing cells. The innovative mouse models proposed in this grant application will generate a novel in vivo system that will help us to clarify the role of Ihh, not only during endochondral bone formation, but also in maintaining endochondral bones. This new information will give us a more secure framework for understanding how Ihh regulates chondrocyte and osteoblast development and function. Such in vivo molecular studies, could, therefore, lead to the discovery of other human skeletal diseases involving the Ihh gene and to novel therapeutic strategies aimed at modulating skeletal anomalies.